Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Cell Biology (ASCB)  

p52SHC (SHC) and GRB2 are adaptor proteins involved in the RAS/MAPK (ERK) pathway mediating signals from cell-surface receptors to various cytoplasmic proteins. To further examine their roles in signal transduction, we studied the translocation of fluorescently-labeled SHC and GRB2 to the cell surface, caused by the activation of ERBB receptors by heregulin (HRG). We simultaneously evaluated activated ERK translocation to the nucleus. Unexpectedly, the translocation dynamics of SHC were sustained when those of GRB2 were transient. The sustained localization of SHC positively correlated with the sustained nuclear localization of ERK, which became more transient after SHC knockdown. SHC-mediated PI3K activation was required to maintain the sustainability of the ERK translocation regulating MEK but not RAF. In cells overexpressing ERBB1, SHC translocation became transient, and the HRG-induced cell fate shifted from a differentiation to a proliferation bias. Our results indicate that SHC and GRB2 functions are not redundant, but that SHC plays the critical role in the temporal regulation of ERK activation.

DOI： 10.1091/mbc.e21-01-0007

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.isci.2021.102724

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MicroRNAs (miRNAs) are cargo carried by extracellular vesicles (EVs) and are associated with cell-cell interactions. The response to the cellular environment, such as disease states, genetic/metabolic changes, or differences in cell type, highly regulates cargo sorting to EVs. However, morphological features during EV formation and secretion involving miRNA loading are unknown. This study developed a new method of EV loading using cell resealing and reconstituted the elementary miRNA-loading processes. Morphology, secretory response, and cellular uptake ability of EVs obtained from intact and resealed HeLa cells were comparable. Exogenously added soluble factors were introduced into multivesicular endosomes (MVEs) and their subsequent secretion to the extracellular region occurred in resealed HeLa cells. In addition, miRNA transport to MVEs and miRNA encapsulation to EVs followed a distinct pathway regulated by RNA-binding proteins, such as Argonaute and Y-box binding protein 1, depending on miRNA types. Our cell-resealing system can analyze disease-specific EVs derived from disease model cells, where pathological cytosol is introduced into cells. Thus, EV formation in resealed cells can be used not only to create a reconstitution system to give mechanistic insight into EV encapsulation but also for applications such as loading various molecules into EVs and identifying disease-specific EV markers.

DOI： 10.1038/s41598-021-82452-5

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RTK-RAS-MAPK systems are major signaling pathways for cell fate decisions. Among the several RTK species, it is known that the transient activation of ERK (MAPK) stimulates cell proliferation, whereas its sustained activation induces cell differentiation. In both instances however, RAS activation is transient, suggesting that the strict temporal regulation of its activity is critical in normal cells. RAS on the cytoplasmic side of the plasma membrane is activated by SOS through the recruitment of GRB2/SOS complex to the RTKs that are phosphorylated after stimulation with growth factors. The adaptor protein GRB2 recognizes phospho-RTKs both directly and indirectly via another adaptor protein, SHC. We here studied the regulation of GRB2 recruitment under the SHC pathway using single-molecule imaging and fluorescence correlation spectroscopy in living cells. We stimulated MCF7 cells with a differentiation factor, heregulin, and observed the translocation, complex formation, and phosphorylation of cell signaling molecules including GRB2 and SHC. Our results suggest a biphasic regulation of the GRB2/SOS-RAS pathway by SHC: At the early stage (<10 min) of stimulation, SHC increased the amplitude of RAS activity by increasing the association sites for the GRB2/SOS complex on the plasma membrane. At the later stage however, SHC suppressed RAS activation and sequestered GRB2 molecules from the membrane through the complex formation in the cytoplasm. The latter mechanism functions additively to other mechanisms of negative feedback regulation of RAS from MEK and/or ERK to complete the transient activation dynamics of RAS.

DOI： 10.2142/biophysico.bppb-v18.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.bbagen.2019.03.015

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Publisher：Springer Nature  

DOI： 10.1038/s41598-019-39973-x

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DOI： 10.1371/journal.pone.0223300

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Phosphatidylinositol-3-phosphate (PI3P) is a lipid that accumulates in the early endosomal membrane, and acts as a scaffold to recruit proteins that contain a PI3P-binding domain, such as the FYVE domain. In this study, we examined the effect of PI3P depletion on the insulin response in rat hepatoma-derived H4IIEC3 cells. We found that insulin treatment induced the transient formation of an actin domain structure, a mesh-like tangled network of actin filaments where phosphorylated Akt, endosomal proteins, and PI3P accumulated. Actin domain formation was repressed by the depletion of PI3P by SAR405, an inhibitor of the class III PI3 kinase, Vps34, by the inhibition of PI3P function by the competitive binding of an excess amount of GST-fused 2xFYVE protein to intracellular PI3P, and by the use of diabetic model cells, in which PI3P was depleted. SAR405 did not affect the phosphorylation level of Akt, and the transcriptional regulation of gluconeogenic and cholesterol synthetic genes after insulin treatment. Interestingly, insulin-induced DNA synthesis was specifically inhibited by SAR405, cytochalasin B, and also in diabetic model cells. These results suggest that PI3P is required for the formation of actin domains, which affected a signaling pathway downstream of Akt associated with DNA synthesis in H4IIEC3 cells.

DOI： 10.1016/j.bbamcr.2019.02.005

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Morphology of Golgi apparatus changes frequently and diversely depending on various cellular conditions and these changes correlate with the balance between membrane inflow and outflow at the Golgi via vesicular transports. In a previous study, we introduced a semi-intact cell system suitable for the reconstitution of morphological changes that organelles undergo as well as the vesicular transport between them. Semi-intact cells are cells that have undergone plasma membrane permeabilization by the cholesterol-dependent pore-forming cytolysin, streptolysin O (SLO). Permeabilization enables the introduction of various molecules into the cells, as well as the substitution of the original cytosol with an exogenously made cytosol prepared from cells in various stages of cell cycle, differentiation, and disease progression. Coupled with a green fluorescent protein(GFP)-visualization technique, this cell-based system enables the analysis of the molecular mechanisms underlying biological processes that are highly dependent on the integrity of the intracellular architecture. In this chapter, we present a variety of reconstitution assays concerning biological reactions pertaining to the Golgi apparatus.

DOI： 10.1007/978-3-030-23173-6_10

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry  

DOI： 10.1039/c7cp05188c

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Cell-based assays have become increasingly important in the preclinical studies for biopharmaceutical products such as specialty peptides, which are of interest owing to their high substrate specificity. However, many of the latter are membrane impermeable and must be physically introduced into cells to evaluate their intracellular activities. We previously developed a "cell-resealing technique" that exploited the temperature-dependent pore-forming activity of the streptococcal toxin, streptolysin O (SLO), that enabled us to introduce various molecules into cells for evaluation of their intracellular activities. In this study, we report a new cell resealing method, the listeriolysin O (LLO)-mediated resealing method, to deliver mid-sized, membrane-impermeable biopharmaceuticals into cells. We found that LLO-type resealing required no exogenous cytosol to repair the injured cell membrane and allowed the specific entry of mid-sized molecules into cells. We use this method to introduce either a membrane-impermeable, small compound (8-OH-cAMP) or specialty peptide (Akt-in), and demonstrated PKA activation or Akt inhibition, respectively. Collectively, the LLO-type resealing method is a user-friendly and reproducible intracellular delivery system for mid-sized membrane-impermeable molecules into cells and for evaluating their intracellular activities.

DOI： 10.1038/s41598-018-20482-2

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Biochemical Society  

DOI： 10.14952/SEIKAGAKU.2018.900433

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/s41598-017-15443-0

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Other Link： http://www.nature.com/articles/s41598-017-15443-0

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Apoptotic death of pancreatic β cells is a major cause of type 2 diabetes mellitus (T2D) progression. Two isoforms of pyruvate kinase, PKM1 and PKM2, have been reported to participate in cell death in several cell types; however, little is known about their causal pathways in pancreatic β-cell death. We examined whether the suppression of PKM1 or PKM2 affects endoplasmic reticulum (ER) stress-induced apoptosis in a pancreatic β-cell line, MIN6, and Beta-TC-6 and found that knockdown of PKM1, but not of PKM2, leads to the induction of ER stress-induced apoptosis in these cells. We also investigated the mechanism by which PKM1 inhibits ER stress-induced apoptosis. We confirmed that PKM1 interacts with A-Raf, an upstream regulator of the MEK/ERK pathway, and that this interaction contributes to MEK1 phosphorylation by A-Raf. PKM1 knockdown suppresses the phosphorylation of MEK, ERK, and caspase-9 (Thr125), which is phosphorylated by the MEK/ERK pathway, thereby inhibiting the cleavage and activation of caspase-9. Thus, PKM1 knockdown activates the caspase-9/caspase-3 pathway under ER stress conditions and leads to apoptosis.

DOI： 10.1016/j.cellsig.2017.07.017

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Cancer cells are under chronic endoplasmic reticulum (ER) stress due to hypoxia, low levels of nutrients, and a high metabolic demand for proliferation. To survive, they constitutively activate the unfolded protein response (UPR). The inositol-requiring protein 1 (IRE1) and protein kinase RNA-like ER kinase (PERK) signaling branches of the UPR have been shown to have cytoprotective roles in cancer cells. UPR-induced autophagy is another prosurvival strategy of cancer cells, possibly to remove misfolded proteins and supply nutrients. However, the mechanisms by which cancer cells exploit the UPR and autophagy machinery to promote survival and the molecules that are essential for these processes remain to be elucidated. Recently, a multipass membrane protein, Yip1A, was shown to function in the activation of IRE1 and in UPR-induced autophagy. In the present study, we explored the possible role of Yip1A in activation of the UPR by cancer cells for their survival, and found that depletion of Yip1A by RNA interference (RNAi) induced apoptotic cell death in HeLa and CaSki cervical cancer cells. Intriguingly, Yip1A was found to activate the IRE1 and PERK pathways of the UPR constitutively in HeLa and CaSki cells. Yip1A mediated the phosphorylation of IRE1 and also engaged in the transcription of PERK. The activation of these signaling pathways upregulated the expression of anti-apoptotic proteins and autophagy-related proteins. These events might enhance resistance to apoptosis and promote cytoprotective autophagy in HeLa and CaSki cells. The present study is the first to uncover a key prosurvival modulator, Yip1A, which coordinates IRE1 signaling with PERK signaling to support the survival of HeLa and CaSki cervical cancer cells.

DOI： 10.1038/cddis.2017.147

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RalGDS is one of the Ras effectors and functions as a guanine nucleotide exchange factor for the small G-protein, Ral, which regulates membrane trafficking and cytoskeletal remodeling. The translocation of RalGDS from the cytoplasm to the plasma membrane is required for Ral activation. In this study, to understand the mechanism of Ras-Ral signaling we performed a single-molecule fluorescence analysis of RalGDS and its functional domains (RBD and REMCDC) on the plasma membranes of living HeLa cells. Increased molecular density of RalGDS and RBD, but not REMCDC, was observed on the plasma membrane after EGF stimulation of the cells to induce Ras activation, suggesting that the translocation of RalGDS involves an interaction between the GTP-bound active form of Ras and the RBD of RalGDS. Whereas the RBD played an important role in increasing the association rate constant between RalGDS and the plasma membrane, the REMCDC domain affected the dissociation rate constant from the membrane, which decreased after Ras activation or the hyperexpression of Ral. The Y64 residue of Ras and clusters of RalGDS molecules were involved in this reduction. From these findings, we infer that Ras activation not merely increases the cell-surface density of RalGDS, but actively stimulates the RalGDS-Ral interaction through a structural change in RalGDS and/or the accumulation of Ral, as well as the GTP-Ras/RalGDS clusters, to induce the full activation of Ral.

DOI： 10.2142/biophysico.14.0_75

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbamcr.2015.05.005

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Forkhead box O1 (FOXO1) is an important target for insulin. It is widely accepted that insulin-induced phosphorylation of FOXO1 by Akt leads to its nuclear exclusion and results in the inhibition of FOXO1-mediated transcription of the gluconeogenic gene phosphoenolpyruvate carboxykinase 1 (PCK1) in hepatocytes. However, many results that contradict this model have accumulated. Here, we provide a new mechanism for insulin-dependent repression of FOXO1-mediated transcription. We showed insulin-induced translocation of endogenous Ser256-phosphorylated FOXO1, which is essential for regulation of FOXO1-mediated transcription, from nuclear speckles to the nuclear periphery. This insulin-dependent translocation of FOXO1 regulated transcriptional repression of PCK1 concomitant with the formation of the FOXO1-euchromatic histone-lysine N-methyltransferase2 (EHMT2) complex and histone modifications of the PCK1 promoter region. Notably, our results suggest that FOXO1 uses nucleoporin 98 kDa NUP98 for this transcriptional regulation. These results provide a new insight into various FOXO1-mediated transcriptional regulation and FOXO1-mediated essential biological pathways.

DOI： 10.1111/gtc.12226

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Rab2A, a small GTPase localizing to the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), regulates COPI-dependent vesicular transport from the ERGIC. Rab2A knockdown inhibited glucose-stimulated insulin secretion and concomitantly enlarged the ERGIC in insulin-secreting cells. Large aggregates of polyubiquitinated proinsulin accumulated in the cytoplasmic vicinity of a unique large spheroidal ERGIC, designated the LUb-ERGIC. Well-known components of ER-associated degradation (ERAD) also accumulated at the LUb-ERGIC, creating a suitable site for ERAD-mediated protein quality control. Moreover, chronically high glucose levels, which induced the enlargement of the LUb-ERGIC and ubiquitinated protein aggregates, impaired Rab2A activity by promoting dissociation from its effector, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in response to poly (ADP-ribosyl)ation of GAPDH. The inactivation of Rab2A relieved glucose-induced ER stress and inhibited ER stress-induced apoptosis. Collectively, these results suggest that Rab2A is a pivotal switch that controls whether insulin should be secreted or degraded at the LUb-ERGIC and Rab2A inactivation ensures alleviation of ER stress and cell survival under chronic glucotoxicity.

DOI： 10.1038/srep06952

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/gtc.12158

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DOI： 10.1038/ncomms3262

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Language：English   Publishing type：Part of collection (book)   Publisher：Nova Science Publishers, Inc.  

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S210_1

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Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.50.510

CiNii Books

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Other Link： https://jlc.jst.go.jp/DN/JALC/10006703508?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)  

The ER-Golgi intermediate compartment (ERGIC) is an organelle through which cargo proteins pass and are being transferred by either anterograde or retrograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus. We examined the effect of 80 different kinase inhibitors on ERGIC morphology and found that rottlerin, a PKCδ inhibitor, induced the dispersion of the perinuclear ERGIC into punctate structures. Rottlerin also delayed anterograde transport of vesicular stomatitis virus G protein (VSVG) from the ER to the Golgi and retrograde transport of cholera toxin from cell surface to the ER via the Golgi. RNA interference revealed that knockdown of PKCδ or ε resulted in the dispersion of the ERGIC, but unexpectedly did not inhibit VSVG and cholera toxin transport. We also found that rottlerin depolarized the mitochondrial membrane potential, as does carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, and demonstrated that a decrease in the intracellular adenosine triphosphate (ATP) levels by rottlerin might underlie the block in transports. These results suggest that PKCδ and ε specifically regulate the morphology of the ERGIC and that the maintenance of ERGIC structure is not necessarily required for anterograde and retrograde transports.

DOI： 10.1016/j.bbamcr.2012.01.007

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Phosphatidylinositol-3-phosphate (PI3P) is a lipid that is enriched specifically in early endosomes. Given that early endosomes containing PI3P act as a microdomain to recruit proteins that contain a PI3P-binding domain (FYVE domain), the equilibrium between the production and degradation of PI3P influences a variety of processes, including endocytosis and signal transduction via endosomes. In the study reported herein, we have developed a novel analytical method to quantify the amount of PI3P in endosomes by introducing a GST-2xFYVE protein probe into semi-intact cells. The GST-2xFYVE probe was targeted specifically to intracellular PI3P-containing endosomes, which retained their small punctate structure, and allowed the semi-quantitative measurement of intracellular PI3P. Using the method, we found that treatment of HeLa cells with H(2)O(2) decreased the amount of PI3P in endosomes in a p38 MAPK-dependent manner. In addition, H(2)O(2) treatment delayed transport through various endocytic pathways, especially post-early endosome transport; the retrograde transport of cholera toxin was especially dependent on the amount of PI3P in endosomes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

DOI： 10.1016/j.bbamcr.2011.01.023

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S5_4

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S145_5

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Language：English   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1242/jcs.063941

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DOI： 10.1128/IAI.01022-09

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S55_1

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S119_1

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DOI： 10.1074/jbc.M109.038869

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DOI： 10.1242/jcs.043414

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Membrane trafficking is an important cellular process that enables the precise localization of membrane proteins. The disturbance of membrane trafficking results in various disease states. To explore systematically the defects in trafficking pathways that cause these disturbances or disease states, we developed an automated high-throughput fluorescence-based imaging system and carried out visual screening for kinase-regulated trafficking pathways of the cation-independent mannose 6-phosphate receptor (CI-M6PR) in HeLa cells. As the result of our visual screening, which examined the effect of kinase inhibitors and a kinase siRNA library, we identified five kinases (CDC42BPB, PRKACA, PRKACG, GSK3 beta and CSNK2A1) that regulate CI-M6PR trafficking. Moreover, we focused on Alzheimer's disease (AD) to study the relationship between the five kinases and a disease state. Notably, two trafficking pathways, which were regulated by PRKACG and GSK3 beta, respectively, induced high levels of secretion of Abeta, the hallmark of AD. In addition, we found that the modulation of GSK3 beta activity affected the microtubule plus end tracking function of cytoplasmic linker protein-associating protein 2 and resulted in the perturbation of BACE1 localization/trafficking and extensive A beta secretion. Our systems provide new approaches for the analysis of spatially-regulated membrane trafficking and related disease states.

DOI： 10.1111/j.1365-2443.2008.01274.x

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DOI： 10.1016/j.bbrc.2008.03.018

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J-GLOBAL

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Recent advances in microscopic techniques have shed light on the roles of specific subcellular structures in the regulation of gene expression. One such structure is the stress granule (SG), which is engaged in stress-triggered translational arrest by sequestering pre-initiation complexes of translation. Recent studies revealed the spatial, compositional, and functional linkage of the SG to the processing body (P-body), another cytoplasmic structure that has been implicated in mRNA degradation and siRNA- or miRNA-mediated gene silencing. In this study, we report that PCBP2, a facilitator of IRES (Internal Ribosomal Entry Site)-mediated translation, is a novel constituent of the SG and P-body. Immunofluorescence studies revealed that while PCBP2 is diffusely distributed throughout the nucleoplasm and the cytoplasm, the protein is enriched in a subset of P-bodies under normal conditions. Upon exposure to heat and arsenic stress, PCBP2 became predominantly accumulated at the SG, but was still present in Dcp1a-positive P-bodies. Live-cell imaging revealed the dynamic association of PCBP2-enriched P-bodies and the SG, and FRAP experiments demonstrated that PCBP2 actively moves in and out of the SG and P-body. Taken together, these results suggest that PCBP2 shuttles between the cytoplasm and the two structures under stress. We propose that PCBP2 may be involved in stress-induced remodeling of mRNP complexes and that it may also play a role in the rapid transition of certain silenced mRNAs into a translationally active state. Additionally, given the property of PCBP2 as a nuclear-cytoplasmic shuttling protein, PCBP2 may play a role in directly targeting nascent mRNPs to specific P-bodies for storage.

DOI： 10.1261/rna.780708

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The mRNA-binding protein CUGBP-1 is a multi-faceted factor, involved in a wide range of biological processes including splicing, translation initiation and mRNA degradation. Here we show that CUGBP-1 is a novel constituent of stress granule (SG), the translational silencing machinery assembled in response to environmental stress. CUGBP-1 was rapidly routed to SGs upon exposure to a variety of environmental stress, and actively shuttles between the nucleus and SGs. The linker domain located between the second and third RNA recognition motifs (RRMs) was found to be essential for the recruitment of CUGBP-1 to SGs. Importantly, we discovered that the linker domain is also required to direct CUGBP-1 to another subcellular structure, perinucleolar compartment (PNC). These results demonstrate the dynamic behavior of CUGBP-1 during stress response and that the linker region, in concert with RRMs, plays a significant role in defining its subcellular localization and dynamics.

DOI： 10.1016/j.yexcr.2007.10.024

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DOI： 10.1016/j.yexcr.2007.10.004

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DOI： 10.1016/j.bbrc.2004.09.173

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Publishing type：Research paper (scientific journal)  

DOI： 10.1091/mbc.E03-11-0822

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Newly synthesized secretory and transmembrane proteins are folded and assembled in the endoplasmic reticulum (ER) where an efficient quality control system operates so that only correctly folded molecules are allowed to move along the secretory pathway. The productive folding process in the ER has been thought to be supported by the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the ER. However, a dilemma has emerged; activation of ATF6, a key regulator of mammalian UPR, requires intracellular transport from the ER to the Golgi apparatus. This suggests that unfolded proteins might be leaked from the ER together with ATF6 in response to ER stress, exhibiting proteotoxicity in the secretory pathway. We show here that ATF6 and correctly folded proteins are transported to the Golgi apparatus via the same route and by the same mechanism under conditions of ER stress, whereas unfolded proteins are retained in the ER. Thus, activation of the UPR is compatible with the quality control in the ER and the ER possesses a remarkable ability to select proteins to be transported in mammalian cells in marked contrast to yeast cells, which actively utilize intracellular traffic to deal with unfolded proteins accumulated in the ER.

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：7 Suppl  

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In mammalian cells, the Golgi apparatus is disassembled at the onset of mitosis and reassembled at the end of mitosis. This disassembly-reassembly is generally believed to be essential for the equal partitioning of Golgi into two daughter cells. For Golgi disassembly, membrane fusion, which is mediated by NSF and p97, needs to be blocked. For the NSF pathway, the tethering of p115-GM130 is disrupted by the mitotic phosphorylation of GM130, resulting in the inhibition of NSF-mediated fusion. In contrast, the p97/p47 pathway does not require p115-GM130 tethering, and its mitotic inhibitory mechanism has been unclear. Now, we have found that p47, which mainly localizes to the nucleus during interphase, is phosphorylated on Serine-140 by Cdc2 at mitosis. The phosphorylated p47 does not bind to Golgi membranes. An in vitro assay shows that this phosphorylation is required for Golgi disassembly. Microinjection of p47(S140A), which is unable to be phosphorylated, allows the cell to keep Golgi stacks during mitosis and has no effect on the equal partitioning of Golgi into two daughter cells, suggesting that Golgi fragmentation-dispersion may not be obligatory for equal partitioning even in mammalian cells.

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NSF and p97 are ATPases required for the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of organelles. NSF uses ATP hydrolysis to dissociate NSF/SNAPs/SNAREs complexes, separating the v- and t-SNAREs, which are then primed for subsequent rounds of fusion. In contrast, p97 does not dissociate the p97/p47/SNARE complex even in the presence of ATP. Now we have identified a novel essential factor for p97/p47-mediated membrane fusion, named VCIP135 (valosin-containing protein [VCP][p97]/p47 complex-interacting protein, p135), and show that it binds to the p97/p47/syntaxin5 complex and dissociates it via p97 catalyzed ATP hydrolysis. In living cells, VCIP135 and p47 are shown to function in Golgi and ER assembly.

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.42.S10_2

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DOI： 10.1083/jcb.149.2.357

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J-GLOBAL

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CiNii Books

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.34.137

CiNii Books

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Other Link： https://jlc.jst.go.jp/DN/JALC/00080668237?from=CiNii

Language：Japanese   Publisher：THE MEMBRANE SOCIETY OF JAPAN  

Membrane fusion plays a crucial role in intracellular vesicular transport, intercellular fusion and viral infectivity. The fusion reaction has been supposed to underlie the following three steps ; (i) specific docking of two membranes, (ii) perturbation of lipid bilayer at fusion point (s) and formingfusion pore (s), (iii) dilation of fusion pore (s). For the intracellular membrane fusion in membrane trafficking pathways, the recent convergence of genetic and biochemical approaches has led to the identification of a number of proteins that involved in specific docking of two membranes (step (i)). However, the details in step (ii) have not been clarified. In this review, I will focus on putative "fusion peptide" of viral fusogenic proteins as an activator of the step (ii). Through the works on the fusion of liposomes mediated by several synthetic peptides that are derived from the fusion peptide of influenza virus hemagglutinin, we found that the fusion is triggered when the peptides become more hydrophobic under fusion-competent conditions, and also found that the common structural features of the fusogenic peptides is not "amphiphilic helical structure". The amphiphilic peptides appearto require other factor (s) to cause fusion. I propose that uneven distribution of the side-chain bulkiness of peptide in ordered structure is important factor for fusion active peptide.

DOI： 10.5360/membrane.19.221

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Other Link： https://jlc.jst.go.jp/DN/JALC/00080699958?from=CiNii

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

Protein-mediated membrane fusion plays a crucial role in the eukaryotic world. Especially, steps such as endocytosis and exocytosis require strongly regulated and coordinated fusion events. The fusion protein, however, is still not identified in spite of increased sophistication of cell-free assay systems for measuring the intracellular fusions because "multi-subunit" protein complex seems to be involved in the fusions. In this review we describe some of our physicochemical approaches for searching and understanding the "fusion machinery" in living cells.

DOI： 10.2142/biophys.32.249

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Other Link： https://jlc.jst.go.jp/DN/JALC/00079021418?from=CiNii

Language：Japanese   Publisher：The Membrane Society of Japan  

Organelles are connected and exchange proteins and lipids each other by vesicular transport, while maintaining their specific morphology and components during interphase. At the onset of mitosis, the balance of vesicular transport between organelles is disturbed, resulting in the drastic change of organelle morphology. To understand the coupling mechanisms between organelle morphology and vesicular transport, we developed the reconstitution systems for the Golgi disassembly, the disruption and reformation of the endoplasmic reticulum (ER), mitotic disruption of ER exit sites, and vesicular ...

DOI： 10.5360/membrane.33.24

CiNii Books

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Language：Japanese  

CiNii Books

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Language：English   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.47.S164_3

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Language：English   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.47.S164_2

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Language：Japanese   Publisher：共立出版  

CiNii Books

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Publisher：日本顕微鏡学会  

DOI： 10.11410/kenbikyo2004.42.143

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Language：Japanese   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.45.S4_1

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Language：Japanese   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.45.153

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Language：Japanese   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.44.S157_4

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Publisher：The Biophysical Society of Japan  

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Language：Japanese   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.44.S157_3

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Language：Japanese   Publisher：バイオテクノロジ-開発技術研究組合  

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Language：Japanese   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.43.S102_1

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Publisher：The Biophysical Society of Japan  

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Language：Japanese   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.42.S80_3

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Language：Japanese   Publisher：The Biophysical Society of Japan  

At the onset of mitosis, each organelle in a mammalian cell must be inherited accurately by two daughter cells. Recent advances in GFP visualization techniques and in genetic approaches have made it possible to understand the dynamic behavior of organelle during their inheritance and to identify the intracellular machinery required for accurate inheritance. Extensive studies have revealed that the inheritance strategies used by each organelle are not stochastic and passive but are rather controlled by the molecules, which are needed for membrane fusion/vesiculation in intracellular membrane...

DOI： 10.2142/biophys.42.116

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Language：Japanese   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.41.S118_3

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Language：Japanese   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.41.S212_1

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Language：Japanese   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.41.S118_4

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Language：Japanese   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.41.S211_4

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Language：Japanese   Publisher：金原一郎記念医学医療振興財団  

DOI： 10.11477/mf.2425902527

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Language：Japanese  

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Other Link： http://search.jamas.or.jp/link/ui/2003123223

Language：Japanese   Publisher：公益社団法人日本生物工学会  

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Language：Japanese   Publisher：秀潤社  

CiNii Books

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Language：Japanese   Publisher：秀潤社  

CiNii Books

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Language：Japanese   Publisher：The Biophysical Society of Japan  

DOI： 10.2142/biophys.39.S47_4

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Language：Japanese   Publisher：The Biophysical Society of Japan  

In mammalian cells, especially in polarized cells, the Golgi apparatus plays a crucial role in the intracellular sorting and directional-transport of proteins and lipids. The dynamic nature of the apparatus is underscored by the cell cycle-dependent redistribustion and coalesce near the restricted pericentriolar and apical locarlzation in polarized cells. Cytoskeletons might be involved in the dynamic nature, the characteristic shape and the intracellular position of the Golgi apparatus. However, the precise mechanisms of the Golgi-positioning and of the maintenance of its shape in polarize...

DOI： 10.2142/biophys.39.29

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese   Publisher：The Biophysical Society of Japan  

CiNii Books

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

CiNii Books

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Language：Japanese   Publisher：The Biophysical Society of Japan  

CiNii Books

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Language：Japanese   Publisher：秀潤社  

CiNii Books

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Language：Japanese   Publisher：日本組織細胞化学会  

DOI： 10.2142/biophys.29.4_211

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Language：Japanese   Publisher：日本組織細胞化学会  

DOI： 10.2142/biophys.29.4_211

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Language：Japanese   Publisher：日本組織細胞化学会  

CiNii Books

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